Bacterial Toxin Research Citations

Materials:

Cholera toxin was purchased from List Biological Laboratory, Inc, California, USA; …

Induction of fluid accumulation by cholera toxin (CT):

Fluid accumulation in mouse intestinal loops in response to CT was performed in ARH1^+/+ and ARH1^-/- mice [28, 39]. After mice were anesthetized, intestine was exteriorized through a midline incision. Two or three intestinal segments of about 4 cm length each were generated by ligation with nylon suture, and 0.2 ml of PBS or PBS containing either 0.5 µg CT or 5 mM 8-Bromo-cAMP were injected into each loop [28]. …

• Product #100B – Cholera Toxin (AZIDE-FREE) from Vibrio cholerae

Indirect immunofluorescence analysis (IFA):

…To resolve the C-terminal topology of TgATPaseP-GC, fresh extracellular parasites were stained with rabbit -HA (1:3000) and mouse -TgSag1 (1:10000) or rabbit -TgGap45 (1:8000) and -TgSag1 (1:10000) antibody combinations before and after permeabilization as described elsewhere (Blume et al. 2009). Briefly, 5 x 104 tachyzoites were released on BSA-coated coverslips and fixed with 4% PFA including 0.05% glutaraldehyde. Permeabilized coverslips were subjected to immunofluorescence assay as indicated above; however, all solutions were substituted to PBS for non-permeabilized staining as principle of the assay as shown in Figure 2B. To find the exact localization of TgATPaseP-GC protein, the IMC was separated from the PM by treating extracellular parasites with -toxin from Clostridium septicum (20 nM, 2 h) (List Biological Laboratories, US) followed by fixation on BSAcoated coverslips. In both cases, the standard secondary antibody staining procedure was performed afterwards, as described for immunofluorescence assay …

Human blood sample processing and in vitro stimulation:

… Human blood was anti-coagulated with 20 units/ml pyrogen-free sodium heparin (American Pharmaceutical Partners, Inc.; Schaumberg, IL). All blood products were kept at room temperature and processed within 4h from collection. For human whole blood assays, neonatal cord blood or adult whole blood (WB) was first diluted with sterile RPMI 1640 medium (Invitrogen; Carlsbad, CA) and 175l of the diluted WB was added to each well of a 96 well U-bottom plate (Becton Dickinson; Franklin Lakes, NJ, USA) containing 75l of RPMI, freshly prepared FIM proteins, or licensed vaccines at 10 the final concentration. The same formulations were used for both in vivo and in vitro assessment, so the FIM2/3 formulation was prepared by mixing dilutions of purified FIM2/3 (200g/ml, List Biological Laboratories, Inc.) in PBS, which at 1/10 human dose would correlate with 5g/250l. …’

• Product #186 – Fimbriae 2/3 from B. pertussis

Intestinal organoid culture and live imaging:

Primary intestinal epithelial organoids were grown as described before⁵⁶. Briefly, the small intestine and colon were flushed and cut into small pieces that were dissociated in PBS containing 2mM EDTA for 30min at 4C. After extensive washing, the isolated crypts were pelleted and mixed with 25l of Matrigel (Corning) and put in a 24-well plate. After polymerization of the Matrigel, complete culture medium containing advanced DMEM/F12 (Gibco) supplemented with B27 supplement (0.02%, Invitrogen), N2 supplement (0.1%, Invitrogen), N-acetylcysteine (0.0025%, Sigma-Aldrich), mouse epidermal growth factor (mEGF; 0.001%, Invitrogen), and conditioned Rspondin and mNoggin medium was added to the wells. Organoids were seeded and imaged in an 8-well chamber (iBidi) for cell death analysis by real time lapse microscopy or in 24-well plates for Western blotting analysis. Cell death was induced with TcdA (1g/ml, Enzo Life Sciences), TcdB (1g/ml, List Laboratories), …

• Product #155 – Toxin B from Clostridium difficile

Toxin preparation:

C. difficile toxin was prepared as previously described (87). Briefly, C. difficile strain r20291 (AB and AB⁺) was cultured in tryptone-yeast extract media for 24 hours in an anaerobic chamber at 37C. Cultures were diluted to an OD that corresponds to 2 10⁷ CFU/ml and spun down; the supernatant was sterilized using 0.22 M filters. Supernatant prepared from toxin-negative (control) and toxin-positive strain was used to stimulate BMDMs at a 1:5 dilution.

Purified TcdB was obtained from List Biological Laboratories (catalog 155) and used at final concentration of 0.5 g/ml, as previously described (7).

• Product #155 – Toxin B from Clostridium difficile

… Bordetella pertussis Adenylate Cyclase Toxin/toxoid (Act) was purchased from Sigma (Cat#A0847, Sigma, St. Louis, MO), and List Biological Laboratories (Cat#188 & 189, List Biological Laboratories Inc. …

• Product #188L – Adenylate Cyclase Toxin, Recombinant from B. pertussis

… in the presence of both Anthrax Lethal Factor (recombinant from B. anthracis) and Anthrax Protective Antigen (recombinant from B. anthracis) of Anthrax (LIST Biological Laboratories INC, USA) in concentrations of 0.5 and 0.1 g/ml, respectively. …

Author did not specify which Lethal Factor (LF) was utilized; List Labs provides the following LF:

• Product #172 – Anthrax Lethal Factor (LF), Recombinant from B. anthracis
• Product #169L – Anthrax Lethal Factor (LF-A), Recombinant from B. anthracis Native Sequence

• Product #171E – Anthrax Protective Antigen (PA), Recombinant from B. anthracis

Toxin neutralization assay (TNA):

An in vitro toxin neutralizing assay (TNA) was carried out with J774A.1 mouse macrophage cell line (ECACC cat no 91051511) as done previously with modifications^24,56. The 96-well flat-bottomed tissue culture plates (Greiner bio one, Germany) seeded with 80 000 macrophages per well in Dulbeccos modified eagle media (DMEM) and 10% foetal calf serum (FCS) were incubated overnight at 37C and 5% CO₂. Test sera were diluted 2-fold (starting dilution; 1:50) in culture medium with PA and LF (List Biological Laboratories Inc., Campbell, USA) at concentrations of 500ng/mL and 400ng/mL respectively. The serum/lethal toxin mixture was incubated for 1h at 37C and 5% CO2 before addition to overnight-cultured cells and incubation for 3h. …

Author did not indicate which specific lethal factor was utilized.  List Labs provides Product #172 (Anthrax Lethal Factor (LF), Recombinant from B. anthracis) and Product #169L (Anthrax Lethal Factor (LF-A), Recombinant from B. anthracis Native Sequence).

• Product #171 – Anthrax Protective Antigen (PA), Recombinant from B. anthracis

Reagents:

… PA (catalog number 171E) and EF (catalog number 178a) were purchased from List Biological Laboratories (Campbell, CA).

ChIP-Seq and data processing:

PBMC were plated at 2 million cells per ml in RPMI containing 10% FBS, 100 units/ml penicillin, and 100g/ml streptomycin. These cells were then treated with 10nM ET (10nM EF and 10nM PA) or 500M 6MB-cAMP for 4.5h and then isolated by centrifugation followed by snap freezing in liquid N₂ as recommended by Active Motif. For each condition, 60 million PBMC were prepared from 3 donors (20 million PBMC per donor). ChIP was performed by Active Motif with antibodies validated for ChIP against CREB (Cell Signaling Tech; catalog number 9197) or against C/EBP (Santa Cruz Biotechnology, catalog number sc-150). After ChIP DNA libraries were constructed, 75-nt sequence reads were generated by Illumina sequencing.

• Product #171E – Anthrax Protective Antigen (PA), Recombinant from B. anthracis
• Product #178A – Anthrax Edema Factor (EF), Recombinant from B. anthracis

… All mice used were between 8 and 12 weeks old. Diphtheria toxin (DT) (List Biological Laboratories, Inc., Campbell, CA) was resuspended in 1X PBS and mice were injected ip with 150 ng/g DT in 100 L of PBS every 24 hours (hrs) for 3 days …

• Product #150 – Diphtheria Toxin, Unnicked, from Corynebacterium diphtheriae